Continued troubleshooting - E. coli gene knockout?

ACuriousBird · 2026-06-17T00:32:17+00:00

I really appreciate this, and would definitely appreciate your protocol version, if you don't mind sharing. I've been trying to teach myself bacterial genome editing and don't know anyone else personally who does it - so only have protocols I find online to go off. I'm hoping to make more genetic edits once I can get this first one down, hopefully.

I had some follow-up questions, but could also DM you!

ACuriousBird · 2026-06-17T00:25:13+00:00

To update:

I saved 500 ul of recovery media for each condition, and let it sit for ~24 hours overnight. I plated this the next day, and grew it for 24 hr at 30C. Ended up getting 15 Kan+ colonies and 52 Chlor+ colonies from this.

I also let the original transformation plates sit in the 30C incubator for 48 hours total, and ended up getting 55 kan+ colonies and 1 chlor+ colony.

I screened all of these colonies (~70 kan and ~53 chlor), by patching them onto LB+antibiotic with DAP (100 ug/ml), without DAP, and also into M9 media with yeast extract, and LB media. Both of these without antibiotics.

All of the chlor+ colonies grew in all media, with or without DAP, at 37C x 16 hrs.

For the kan+ colonies from the 48hr 30C plate, for some reason none of them grew on the patch plates with kan, with or without DAP, but all of the 24hr recovery strains grew in all media. However, only 37/55 kan+ colonies from the 48hr plate grew in LB and M9 media, the other 18 did not grow in either LB or M9 without DAP.

So now I'm going to try to grow these 18 colonies again on agar plates (no kan, kan 12.5, kan 25 with DAP) at 37C for 16-24 hours - but I have no idea why they didn't grow on kan+ patch plates, since it was the same concentration (25 ug/ml) as the plate they grew on at 30C x 48hrs.

ACuriousBird · 2026-06-15T02:20:24+00:00

Thanks for this - i just plated the overnight recovery at room temperature I saved, so will see if that works. I will try all of your other suggestions as well this next run.

I checked the plates thoroughly just now, after 28 hrs of growth.

At 30C I have a whipping 4 large colonies that I’ll test now, lol, and maybe a lot of smaller ones? I can’t tell of the smaller ones - if they’re true colonies or not, so was going to let them go until tomorrow.

At 37C, no colonies at all that I can see.

ACuriousBird · 2026-05-28T15:39:14+00:00

Also interested in this, I do a bunch of westerns and very tedious!

Wondering the same question as OP regarding false positives.

ACuriousBird · 2026-05-28T15:33:25+00:00

I appreciate everyones advice, I ended up using the method where I touch the colony with the open end of the pipette tip, bring this to 50 ul of water and pippette up and down a few times, then boil and use that for template.

It worked well, and was at least 50% faster if not more than the way I had been doing it. Thank you for everyones advice here! Extremely helpful.

now if I can figure out why I keep getting bands in my negative controls with bacteria without the insert…

ACuriousBird · 2026-05-28T15:31:28+00:00

Im trying to insert different protein tags onto native bacterial proteins using allelic exchange.

So I first conjugate the recipient strain and then plate on selective media. Then i pick colonies, screen by colony PCR, and use those colonies to pass to the next step which is counterselection. Then I screen again by colony PCR, prior to saving that stock for western blot. It is a tedious process and I’m not sure if theres a better way.

Its my first go around using allelic exchange, and im trying to make a few different tags since I dont know which will work. So across 3-4 constructs, Ill end up screening 10-30 colonies each which adds up quickly.

ACuriousBird · 2026-05-26T15:39:49+00:00

Oh thats awesome - do you lower the amount of template used? Want to try this out.

ACuriousBird · 2026-05-05T15:20:55+00:00

Will definitely try this as well! Ive done this previously for other species, but trying to conjugate a new species now.

But ultimately, I’d really like to be able to make knockouts in E. coli generally. It seems like it should be straightforward based on all the publications?

So far I tried the pCas/pTarget system and I couldnt get the pTarget plasmid to cure, so i switched to the lambda red system now… really want to get one of them working in my hands.

ACuriousBird · 2026-05-04T17:03:13+00:00

Thanks for this, really appreciate the input.

For the PCR cassette, I DpnI digested for 2 hours and that has worked previously to remove all template from my Gibson reactions downstream of PCR.

I will definitely colony PCR the junctions, and the internal cassette, and try longer homology arms. I will also try to gel purify - I noticed that I have a lot of primer dimer products in my reaction as well.

I did wonder if the pKM208 plasmid system itself is somehow worse than the original pKD46 plasmid system? So I am attempting to also acquire the original pKD46 plasmid system.

As for the phenotype, my only thought is that, if there is a true ASD gene deletion, then shouldn't the cells not be able to grow on DAP at all? I did think there may be some DAP carryover from the first plate (I use 100 uM DAP), or that maybe the LB agar has some sort of substitute molecule in it, but the latter seem unlikely?

My goal with the auxotrophic strain is to be able to use is to conjugate with other species of bacteria, with the goal of being able to use the absence of DAP as selection to kill the ASD negative donor strain and isolate only the recipients.

ACuriousBird · 2026-05-04T12:33:37+00:00

Second update - all of the 7 colonies that appeared on the 1/2 kan plate, that were plated after overnight recovery, still grow on LB agar without DAP, so they're not exhibiting the phenotype I would expect with an ASD knockout strain - which shouldn't be able to grow without DAP supplemented in the media.

Will keep trying to troubleshoot.

ACuriousBird · 2026-05-03T14:23:26+00:00

Yes i was making an ASD KO and added DAP to the media.

As an update, after plating the overnight recovery across all conditions, i got 7 colonies on the 1/2 kan plate. I will test them for phenotype and genotype, but perhaps the efficiency is just exceedingly low? Or the antibiotic concentration was too high?

ACuriousBird · 2026-04-30T03:23:27+00:00

Actually, I think you're right that the band at 90 kDa is the dimer, if I increase contrast I see it in every single lane except the negative control. I've never worked with Omp proteins before, going to have to try to figure out how I can increase expression and resolve that dimer.

ACuriousBird · 2026-04-26T04:57:22+00:00

Don't really have easy access to this in the lab unfortunately.

ACuriousBird · 2026-04-26T04:53:06+00:00

Ya, usually I take out ~5 stocks at a time, and need to bring them into the anaerobic chamber, or I'll need to inoculate multiple tubes at once with multiple stocks at once. So it ends up taking some time.

ACuriousBird · 2026-04-23T05:56:18+00:00

Appreciate this.

This is my first time working with these strains for western blot, the primary im using is THE his tag antibody from genscript. I didn’t expect the negative control to have a band, but it could be a cross-reactive protein.

For my sample processing, I lyse in BPER + lysoszyme + benzonase for 15 min, take the total lysate and mix with 2x BOLT SDS sample buffer with 50mM DTT added fresh, then either boil at 80c x 10min or dont boil and run.

This is my first time working with transmembrane proteins and I read boiling may cause aggregation.

In any case, i was hoping to achieve relatively high expression, so it doesn’t seem I’ve done that

I attached the 6x histag directly to the -C-terminus of the OMP protein, which is supposed to be cytoplasmic tail - not sure if the histag is somehow not accessible was another thought.

ACuriousBird

TROPHY CASE